Adherent cells are always required to attach to some surface carrier for growth. T-Flasks and roller bottles are the most commonly used devices for these types of cell cultures in research use. Roller bottles have also been widely adopted for production use. However due to limited surface area available in each bottle, the use of roller bottle culture is labor intensive for production use. It is also lack of controllability and therefore gradually being replaced by other culture methods for adherent cell cultures such as methods using microcarrier and packed bed bioreactors which are commonly cultured in three dimensional (3D) carriers. However, many products, such as stem cells are preferentially required to be produced in two dimensional growth or as a monolayer. The simplicity of cell detachment and recovery of cells to meet the regulatory requirement is another reason to use the two dimensional growth culturing method. The debris of 3D fibrous carriers is not acceptable for cell therapy use. Therefore 2D culture method is still commonly used. The other reason is that most bioproducts derived from cell culture have been developed on 2D culture vessels such as the T-flask, and are ready to comply with regulatory requirements by remaining in the same 2D culture vessel for large scale production. To scale up from T-flask and roller bottle, the multi-tray bioreactors are the most commonly used systems available on the market today including Corning's Cellstack®, Hyperstack® and Thermo's Cell Factory® for static cultures.
For static cultures the multi-tray vessel with multiple parallel trays stacked as a vessel such as a 40 tray Cell Factory® can have a surface area of 25280 cm2 in one vessel. However, the vessel can become very heavy, bulky, and labor intensive. Thermo therefore have developed automatic manipulators to perform the filling and emptying processes for the static culture. The manipulator employs a robotic arm to perform the manual operation by turning the vessel about two axes in three dimensions for filling and emptying. Then they need to use the cart to transport the filled vessel to an incubator for culturing. After culturing they need further to transport it to the manipulator and shaker for enzymatic treatment and detaching the cells. The entire operation is complicated, the footprint is large, the entire process is not integrated and still subject to the risk of contamination. Above all the system is extremely expensive (>$700 k) for manipulator, shaker, incubator and cart. This disclosure provides a unique solution enabling to perform the entire culturing process including filling, seeding, culturing, medium exchange, infection, transfection, sampling, cell detachment and harvesting automatically using the multi-tray culture vessels all in one closed system. US2010210016, US20060019388 and US20150017711A1 taught to rotate the culture chamber in two axes to perform each culturing process automatically. US 20090298164 and US20150017711A1 taught to use multi-layer vessel to perform static culture. US20120086657 and US20150017711A1 taught to apply system configuration to perform automation. None of the teachings however taught to use active gassing to the multi-tray 2D flat surface culture vessels for static culture and apply an integrated vessel to rotate only in one axis and use the same vessel as an incubator to perform the entire culturing process aforementioned all in one closed system.
For dynamic culture, Pall's Xpansion® is a multi-plate bioreactor using an agitated system to circulate the medium with constant flow over the surface plates and oxygenates by the diffusion of oxygen through silicon tubing. The efficiency of oxygenation is limited. Like Thermo's automated AMCF system, Xpansion® requires separate incubator for culturing and harvest station for cell detachment and recovery. Again it is not integrated and subject to the risk of contamination. Above all the system is very expensive. This disclosure alleviates all the limitation and complexity. It applies the most efficient and simple oxygenation method similar to roller bottle system and enables to make the system very simple. Above all, it is an integrated system and able to perform the entire culturing process all in one close system automatically.